Jan 05, 2022
Pharyngeal Pumping Assay
- 1Imperial College London
- Behavioural Genomics
Protocol Citation: Thomas J O'Brien 2022. Pharyngeal Pumping Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.b3hiqj4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 05, 2022
Last Modified: January 05, 2022
Protocol Integer ID: 56586
Abstract
The pharynx is a is a neuromuscular pump found at the anterior end of the alimentary tract, consisting of 20 muscles and 20 neurons. A proper feeding rate in worms is coordinated by the precise timing of pharyngeal movements, with one complete cycle of synchronous contraction and relaxation of the corpus and terminal bulb termed a “pump”. A simple way to measure C. elegans feeding is to count how many times worms pump in a minute (pumps per minute). Movement of the grinder (in the terminal bulb) can easily be observed using a stereomicroscope, and because cycles of contraction/relaxation are synchronised along the pharynx, pumps per minute can be measured simply by counting grinder movements.
Materials
Consumables per worm strain:
- 4 x 90 mm petri dish
- 5 x 60 mm petri dish
Reagents for bleaching:
- Sodium hypochlorite, 5% Chlorine (Fisher Scientific, Catalog #419550010)
- 1 M Sodium Hydroxide (Merck Millipore, Catalog #1091371000)
Reagents for preparing NGM agar:
| A | B | C | |
| Pre-autoclave | For 1L: | For 500mL: | |
| Sodium chloride | 3g | 1.5g | |
| BioAgar | 17g | 8.5g | |
| Bacto Peptone | 2.5g | 1.25g | |
| Sterile water | 975mL | 487.5mL |
| A | B | C | |
| Post-autoclave | Fo 1L: | For: 500mL | |
| 5mg/mL cholesterol (store at 4°C and away from light) | 1mL | 500uL | |
| 1M CaCl2 | 1mL | 500uL | |
| 1M MgSO4 | 1mL | 500uL | |
| 1M KPO4 | 25mL | 12.5mL |
Pick L4 worms for bleaching (Day 1)
Pick L4 worms for bleaching (Day 1)
Using an eyelash pick, pick 10 x L4s of each worm strain to be assayed onto 2 x 90 mm plates (pre-seeded with OP50) and incubate at 20 °C
Pour 60 mm NGM agar plates (Day 2)
Pour 60 mm NGM agar plates (Day 2)
Prepare and pour the following number of NGM plates per worm strain to be studied:
- 5 x 60 mm (15 mL agar per plate)
- 2 x 90 mm (35 mL agar per plate)
Therefore make 150 mL NGM per strain
To ensure accuracy when weighing out media components it is recommended that you do not prepare agar in volumes less than 250 mL at a time!
In the laminar flow hood, pour 5 x 60 mm and 2 x 90 mm plates per worm strain
Protocol
NAME
Plate PouringCREATED BY
Priota Islam
Once plates have dried, store agar-side up in an airtight container in the cold room
Dry/Seed 90 mm plates and bleach worms (Day 5)
Dry/Seed 90 mm plates and bleach worms (Day 5)
Dry 2 x 90 mm plates per worm strain in the cabinet dryer (setting 2) for 1.5 hours
Seed the 90 mm plates with OP50 and leave to dry overnight at room temperature
Bleach synchronise worms (picked in Step 1) and store eggs/larvae in a 15 mL falcon tube on the rotator that is constantly spinning at 20 °C until feeding
Protocol
NAME
Bleach synchronisation of C. elegansCREATED BY
Ida Barlow
Refeed L1s (Day 8)
Refeed L1s (Day 8)
Refeed bleach synchronised L1s
At 16:50, spin L1s bleached in step 4 using centrifuge program 1 (2500 rpm, 2 mins)
Using a plastic pipette remove the supernatent, taking care to not disturb the worm pellet, and leave ~1 mL of M9 in the tube
Gently flick the falcon tube to resuspend the L1s
Using a glass pipette, drop 1 small droplet around the edges (off food) of the pre-seeded 90 mm plates (prepared in Step 3)
It is recommended that you check the density of worms in the droplet down a microscope and add another if necessary
Place plates agar-side down in the 20 °C incubator to allow droplets to dry (~5-10 mins), then invert plates and allow to grow for 2.5 days
Dry and seed 60 mm plates (Day 10)
Dry and seed 60 mm plates (Day 10)
In the morning, take the 60 mm plates from the cold room, and weigh three random plates without their lids
Dry plates in in cabinet dryer (setting 2) with lids off until they have lost 3-5% of their weight (approx 2 hours)
Aseptically prepare 1:10 dilution of OP50 in M9 in a 15 mL falcon tube:
1 mL OP50
9 mL M9
Working by a flame, dispense 30 µL of the dilute OP50 into the centre of each 60 mm plate and allow droplets to dry at room temp
Ensure that you do not touch the agar with the pipette tip and that plates are kept on a flat surface so you get a nice, even circle of food once dried
Once the food has dried, leave plates agar-side up at room temp overnight
Assaying rate of pharyngeal pumping (Day 11)
Assaying rate of pharyngeal pumping (Day 11)
Label 5 x 60 mm plates seeded with 30 µL OP50 (around the edge, so not to obscure the bacterial lawn) per worm strain
At 09:00, use an eyelash-pick to transfer 10 x young adult worms (prepared in Step 5) onto the edge of each 60 mm plate (away from food)
This yields a total of 50 worms per strain
Incubate plates, agar-side up, at 20 °C for 1 hour
This allows worms to crawl to the bacterial lawn and start foraging again
Working one plate at a time, place a 60 mm plate upon a dissecting stereo microscope at the highest magnification possible and bring the worms/bacterial lawn to focus
Working on a single worm at a time, identify the grinder (in the terminal bulb of the head) of each worm and ensure this is clearly in focus
See: http://www.wormbook.org/chapters/www_measurepharyngeal/measurepharyngeal.html for detailed images/diagrams of pharynx and terminal bulb
Use a tally counter to count the grinder movements of a worm over a 20 second period
Repeat this 3 times per worm and record the result as an average of the three counts
Go clockwise around the bacterial lawn, and repeat the steps above for each worm on the 60 mm plate. Then repeat for each 60 mm plate remaining in the incubator
Calculate pharyngeal pumps per minute of each strain:
pumps per minute = pumps per 20 seconds * 3